Figure 1: CD81 target protein structure.

CD81 Target Introduction

Protein Function

• CD81 is a member of the tetraspanin superfamily (TM4SF) and a four-transmembrane protein. It can form complexes with other tetraspanin proteins, integrins, co-receptors, and MHC class I or II molecules, affecting the adhesion, morphology, activation, proliferation, and differentiation of B and T cells.
• In hepatocytes, it acts as a receptor for hepatitis C virus (HCV), and the formation of the CLDN1-CD81 receptor complex is crucial for HCV entry into host cells.
• In T cells, it is involved in SAMHD1 inhibition of HIV-1 replication. CD81 regulates the subcellular localization of SAMHD1 and degrades it through the proteasome pathway, thereby controlling the intracellular dNTP levels.
• CD9, CD63, CD81, etc., are often used as exosome surface markers.

Protein Expression

• CD81 is expressed in B cells, monocytes, macrophages, naive CD4+ T cells, and memory CD4+ T cells.

Protein Localization

• CD81 is localized to the cell membrane and basal membrane.

Figure 2: CD81 ICC experimental result image, Anti-CD81 antibody [EPR21916] (ab219209). Green: CD81, Red: Tubulin, Blue: DAPI.

Isoforms & Post-translational modifications

• Human (P60033): 25 kDa (predicted)
• Mouse (P35762): 25 kDa (predicted)
• Rat (Q62745): 25 kDa (predicted)
• No glycosylation modification. Possible presence of palmitoylation.

WB experiment tips

Precautions

• When detecting CD81 in extracellular vesicles, the protein abundance of CD81 is significant. There may be no signal if the CD81 content in the extracted extracellular vesicles is low. Please refer to the references for more extracellular vesicle extraction protocols and research guidelines [1,2].
• We strongly recommend using a positive control (such as U87-MG).
• The predicted molecular weight of the protein is about 25 kDa, and the actual detected molecular weight is about 20 kDa. Therefore, please follow the precautions for small protein experiments during electrophoresis and transfer processes.

Positive controls

• Human Ramos whole cell lysate, U87-MG whole cell lysate
• Rat PC-12 cell lysate
• Mouse Raw264.7 cell lysate

Example of results

Figure 3: WB-Anti CD81 antibody [EPR4244] (ab109201).

Lane 1: Raw264.7 whole cell lysate
Lane 2: C2C12 whole cell lysate
Lane 3: Mouse brain tissue lysate
Lane 4: Mouse heart tissue lysate
Lane 5: Huh7 whole cell lysate
Lane 6: HL-60 whole cell lysate
Lane 7: HCT116 whole cell lysate
Lane 8: LNCaP whole cell lysate
Lane 9: U-87MG whole cell lysate
Lane 10: HeLa whole cell lysate
Lane 11: A549 whole cell lysate
Lane 12: 293T whole cell lysate

Primary antibod y: Anti-CD81 antibody [EPR4244] (ab109201), diluted 1/1000.
Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051), diluted 1/20000.
Predicted band size: 26 kDa
Detected band size: 25 kDa

Figure 4: WB- Anti-CD81 antibody [EPR4244] (ab109201).

Lane 1: 10 µg Ramos cell lysate
Lane 2: 10 µg U87-MG cell lysate
Lane 3: 10 µg PC-12 cell lysate
Lane 4: 10 µg Raw264.7 cell lysate

Primary antibody: Anti-CD81 antibody [EPR4244] (ab109201), diluted at 1/2000 concentration.
Secondary antibody: Peroxidase-conjugated goat anti-rabbit antibody, diluted at 1/1000 concentration.
Predicted band size: 26 kDa
Detected band size: 20 kDa

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex proteinase inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.
   We recommend using a positive control.

Electrophoresis:

1. For target proteins with smaller molecular weights, we recommend using a 15% concentration separation gel for electrophoresis.

Transfer:

1. We recommend using a 0.22 μm PVDF membrane.
2. We recommend using 20% methanol in the transfer buffer.
3. We recommend using Coomassie Brilliant Blue staining after transfer to confirm the success of the transfer.

References

1. Emma J K Kowal, Dmitry Ter-Ovanesyan, Aviv Regev, etc. Extracellular Vesicle Isolation and Analysis by Western Blotting. Methods Mol Biol. 2017;1660:143-152. doi: 10.1007/978-1-4939-7253-1_12
2. Clotilde Théry, Kenneth W Witwer, Elena Aikawa, etc. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018 Nov 23;7(1):1535750. doi: 10.1080/20013078.2018.1535750.
3. Yoshito Takeda, Isao Tachibana, Kenji Miyado, etc. Tetraspanins CD9 and CD81 function to prevent the fusion of mononuclear phagocytes. J Cell Biol. 2003 Jun 9;161(5):945-56. doi: 10.1083/jcb.200212031.
4. Menno C van Zelm, Julie Smet, Brigitte Adams, etc. CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency. J Clin Invest. 2010 Apr;120(4):1265-74. doi: 10.1172/JCI39748. Epub 2010 Mar 8.
5. Helen J Harris, Christopher Davis, Jonathan G L Mullins, etc. Claudin association with CD81 defines hepatitis C virus entry. J Biol Chem. 2010 Jul 2;285(27):21092-102. doi: 10.1074/jbc.M110.104836. Epub 2010 Apr 7.
6. Vera Rocha-Perugini, Henar Suárez, Susana Álvarez, etc. CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels. Nat Microbiol. 2017 Nov;2(11):1513-1522. doi: 10.1038/s41564-017-0019-0. Epub 2017 Sep 4.
7. Yandong Li, Shijun Yu, Li Li, etc. KLF4-mediated upregulation of CD9 and CD81 suppresses hepatocellular carcinoma development via JNK signaling. Cell Death Dis. 2020 Apr 29;11(4):299. doi: 10.1038/s41419-020-2479-z.

PMID: 28828654, 30637094